Adult Chondrogenesis and Spontaneous Cartilage Repair in the Skate, Leucoraja Erinacea Aleksandra Marconi1, Amy Hancock-Ronemus2,3, J Andrew Gillis1,3*
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RESEARCH ARTICLE Adult chondrogenesis and spontaneous cartilage repair in the skate, Leucoraja erinacea Aleksandra Marconi1, Amy Hancock-Ronemus2,3, J Andrew Gillis1,3* 1Department of Zoology, University of Cambridge, Cambridge, United Kingdom; 2Charles River Laboratories, Wilmington, Massachusetts, United States; 3Marine Biological Laboratory, Woods Hole, Massachusetts, United States Abstract Mammalian articular cartilage is an avascular tissue with poor capacity for spontaneous repair. Here, we show that embryonic development of cartilage in the skate (Leucoraja erinacea) mirrors that of mammals, with developing chondrocytes co-expressing genes encoding the transcription factors Sox5, Sox6 and Sox9. However, in skate, transcriptional features of developing cartilage persist into adulthood, both in peripheral chondrocytes and in cells of the fibrous perichondrium that ensheaths the skeleton. Using pulse-chase label retention experiments and multiplexed in situ hybridization, we identify a population of cycling Sox5/6/9+ perichondral progenitor cells that generate new cartilage during adult growth, and we show that persistence of chondrogenesis in adult skates correlates with ability to spontaneously repair cartilage injuries. Skates therefore offer a unique model for adult chondrogenesis and cartilage repair and may serve as inspiration for novel cell-based therapies for skeletal pathologies, such as osteoarthritis. Introduction Hyaline cartilage is a skeletal tissue that consists of a single cell type (the chondrocyte) embedded *For correspondence: [email protected] within a homogeneous, collagenous extracellular matrix (reviewed in Gillis, 2018). In mammals, hya- line cartilage is predominantly an embryonic tissue, making up the anlage of the axial (chondrocra- Competing interests: The nial, vertebral and rib) and appendicular (limb) endoskeleton. The vast majority of mammalian authors declare that no hyaline cartilage is replaced by bone during the process of endochondral ossification, with cartilage competing interests exist. persisting temporarily in epiphyseal growth plates, and permanently at relatively few sites within the Funding: See page 22 adult skeleton (e.g. in joints, as articular cartilage – Decker, 2017). In juvenile mammals, growth of Received: 07 November 2019 articular cartilage occurs by cellular rearrangement and increases in chondrocyte volume Accepted: 21 April 2020 (Decker et al., 2017), and by appositional recruitment of new chondrocytes from a superficial popu- Published: 12 May 2020 lation of slow-cycling chondroprogenitor cells (Hayes et al., 2001; Dowthwaite et al., 2004; Karlsson et al., 2009; Williams et al., 2010; Kozhemyakina et al., 2015). However, evidence for Reviewing editor: Alejandro the presence of chondroprogenitor cells in adult articular cartilage is scant, and this – combined with Sa´nchez Alvarado, Stowers Institute for Medical Research, the avascular nature of the tissue – may account for why mammalian articular cartilage cannot heal United States spontaneously following injury (Hunziker, 1999). Chondrichthyans (cartilaginous fishes – sharks, skates, rays and holocephalans), on the other Copyright Marconi et al. This hand, possess an endoskeleton that is composed largely of hyaline cartilage, and that remains carti- article is distributed under the laginous throughout life. Though chondrichthyans reinforce their endoskeleton with a superficial terms of the Creative Commons Attribution License, which layer of calcified cartilage (in the form of small mineralized plates called ‘tesserae’ – Dean and Sum- permits unrestricted use and mers, 2006), the core of their endoskeletal elements persists as hyaline cartilage and does not redistribution provided that the undergo endochondral ossification. Like many fishes (and unlike mammals), chondrichthyans also original author and source are exhibit an indeterminate type of growth, with a continued (albeit slow) increase in size through adult- credited. hood (Dutta, 1994; McDowall, 1994; Frisk and Miller, 2006). It therefore stands to reason that, in Marconi et al. eLife 2020;9:e53414. DOI: https://doi.org/10.7554/eLife.53414 1 of 26 Research article Developmental Biology Stem Cells and Regenerative Medicine eLife digest For our joints to move around freely, they are lubricated with cartilage. In growing mammals, this tissue is continuously made by the body. But, by adulthood, this cartilage will have been almost entirely replaced by bone. It is also difficult for adult bodies to replenish what cartilage does remain – such as that in the joints. When growing new cartilage, the body uses so-called progenitor cells, which have the ability to turn into different cell types. Progenitor cells are recruited to the joints, where they transform into cartilage cells called chondrocytes, which generate new cartilage. But adults lack these progenitor cells, leaving them unfit to heal damaged cartilage after injury or diseases like osteoarthritis. In contrast, certain groups of fishes, such as skates, sharks and rays, produce cartilage throughout their life — indeed their whole skeleton is made of cartilage. So, what is the difference between these cartilaginous fishes and mammals? Why can they generate cartilage throughout their lives, while humans are unable to? And does this mean that these adult fish are better at healing injured cartilage? Marconi et al. used skates (Leucoraja erinacea) to study how cartilage develops, grows and heals in a cartilaginous fish. Progenitor cells were found in a layer that wraps around the cartilage skeleton (called the perichondrium). These cells were also shown to activate genes that control cartilage development. By labelling these progenitor cells, their presence and movements could be tracked around the fish. Marconi et al. found progenitor cells in adult skates that were able to generate chondrocytes. Skates were also shown to spontaneously repair damaged cartilage in experiments where cartilage was injured. Marconi et al. have identified the skate as a new animal model for studying cartilage growth and repair. Studying the mechanisms that skate progenitor cells use for generating cartilage could lead to improvements in current therapies used for repairing cartilage in the joints. chondrichthyans, skeletal tissues may possess a persistent pool of chondroprogenitor cells to facili- tate continued growth of their cartilaginous endoskeleton throughout adulthood, and that such cells (if present) could also impart the endoskeleton with an ability to undergo spontaneous repair follow- ing injury. However, basic mechanisms of hyaline cartilage development, growth and repair in chon- drichthyans remain largely unexplored. Here, we characterize the development and growth of the cartilaginous endoskeleton of a chon- drichthyan, the little skate (Leucoraja erinacea), from embryonic development to adulthood. We demonstrate conservation of fundamental cellular and molecular characteristics of cartilage develop- ment between chondrichthyans and mammals, and we identify unique features of adult skate carti- lage that contribute to its continued growth through adulthood. We further show that skates can repair surgically induced partial-thickness cartilage injuries, highlighting this system as a unique ani- mal model for adult chondrogenesis and spontaneous hyaline cartilage repair. Results The metapterygium of the skate, Leucoraja erinacea The pectoral fin endoskeleton of jawed vertebrates consisted ancestrally of three basal cartilages – from anterior to posterior, the propterygium, mesopterygium and metapterygium – and a series of articulating distal radials (Davis et al., 2004). Among extant jawed vertebrates, this ancestral ‘tri- basal’ condition has been retained in the pectoral fins of chondrichthyans and non-teleost ray-finned fishes (e.g. in sturgeon, gar and bichir), but has been reduced in tetrapods and teleosts, to include only derivatives of the metapterygial and pro-/mesopterygial components, respectively (Davis, 2013). Our study focused on the metapterygium of the skate (Figure 1), as this element is relatively large, reliably identifiable across all embryonic and post-embryonic stages and easily acces- sible for surgical manipulation. Marconi et al. eLife 2020;9:e53414. DOI: https://doi.org/10.7554/eLife.53414 2 of 26 Research article Developmental Biology Stem Cells and Regenerative Medicine Figure 1. The metapterygium of the little skate, Leucoraja erinacea. A radiograph reveals the skeletal anatomy of an adult skate. The metapterygium (false colored red in i) is the caudalmost basal fin cartilage. The plane of section through the metapterygium used for Figures 2–6 is indicated with a dashed line and x. Scale bar = 5 cm. Embryonic development and growth of cartilage in the skate metapterygium In mammals, early cartilage development is marked by the accumulation of preskeletal mesenchyme into a ‘condensation’ at the site of future chondrogenesis (Hall and Miyake, 2000). Cells within this condensation begin to secrete cartilage extracellular matrix (ECM) components and undergo overt differentiation into chondrocytes. To investigate the early development of cartilage in the skate, we prepared a histological series of skate metapterygia from embryonic stage (S) 30 through to hatch- ing and used a modified Masson’s trichrome stain to visualize condensation, differentiation and ECM secretion. At S30, the presumptive metapterygium exists as condensed mesenchyme, with pericellular Light Green staining (which appears